Download Tumour necrosis factor-a (-308) gene polymorphism in obstructive sleep apnoea–hypopnoea syndrome

Eur Respir J 2005; 26: 673–678
DOI: 10.1183/09031936.05.00130804
CopyrightßERS Journals Ltd 2005
Tumour necrosis factor-a (-308) gene
polymorphism in obstructive sleep
apnoea–hypopnoea syndrome
R.L. Riha*, P. Brander*, M. Vennelle*, N. McArdle*, S.M. Kerr#,
N.H. Anderson" and N.J. Douglas*
ABSTRACT: Patients with obstructive sleep apnoea–hypopnoea syndrome (OSAHS) have
elevated circulating levels of tumour necrosis factor (TNF)-a. The hypothesis in this study was that
OSAHS might be associated with the TNF-a (-308A) gene polymorphism, which results in
increased TNF-a production. This hypothesis was examined in OSAHS patients, their siblings and
population controls.
A total of 206 subjects were recruited. All underwent sleep studies and clinical review, and were
subsequently classified as having OSAHS or not depending on apnoea–hypopnoea frequency,
sex, age and symptoms. All subjects had blood collected and genotyping was performed on DNA
extracted from peripheral leukocytes. Some 192 random UK blood donors were used as
population controls.
The results demonstrated a significant association for TNF-a (-308A) allele carriage with OSAHS
(OR51.8; 95% Confidence interval: 1.18–2.75) when compared with population controls. Siblings
with OSAHS were significantly more likely to carry the TNF-a (-308A) allele. In addition, 21 pairs of
male siblings discordant for carriage of the -308A allele showed a significant level of discordance
for the OSAHS phenotype.
In conclusion, this study demonstrates an association of tumour necrosis factor-a (-308A)
carriage with obstructive sleep apnoea–hypopnoea syndrome, suggesting that inflammation may
be implicated in the pathogenesis of this condition.
AFFILIATIONS
*Respiratory Medicine, and
"
Public Health Sciences, University
of Edinburgh, and
#
Genetics Core, Wellcome Trust
Clinical Research Facility, Edinburgh,
UK.
KEYWORDS: Genetics, obstructive sleep apnoea, tumour necrosis factor-a
SUPPORT STATEMENT
R.L. Riha was the recipient of an
Australian Federation of University
Women (AFUW) Fellowship,
European Respiratory Society (ERS)
Long-Term Training Fellowship and
Helen Bearpark Scholarship whilst
undertaking this work. P. Brander was
the recipient of a scholarship from
the Finnish Anti-Tuberculosis
Association Foundation.
bstructive sleep apnoea–hypopnoea syndrome (OSAHS) is the second most
common respiratory condition, affecting
,0.3–4% of the middle-aged population. It is
defined on the basis of symptoms of daytime
sleepiness and objective measures of disordered
breathing during sleep [1, 2]. OSAHS has a
familial component, independent of obesity [3],
and there is increasing evidence that OSAHS is
associated with inflammation. Raised levels of Creactive protein, hypercytokinaemia and tumour
necrosis factor (TNF)-a have been reported in
OSAHS [4–6]. The raised serum TNF-a level is
independent of obesity and correlates with
systemic blood pressure [5, 7, 8]. Furthermore,
TNF-a also promotes sleepiness, the major
feature of OSAHS [9].
O
position -308 in the promoter region [10, 11]. It
was thus hypothesised that OSAHS might be
associated with the TNF-a (-308A) gene polymorphism. This was tested in OSAHS patients,
their siblings and population controls.
METHODS
A case-control study, based on the recruitment of
patients with OSAHS and their available siblings,
was undertaken between January 1997 and
December 2002 at the Scottish Sleep Centre
(Edinburgh, UK). A total of 192 random anonymous UK blood donors were used as additional
population controls. Approval for the study was
obtained from the local research ethics committee
(Edinburgh, UK).
Increased production of TNF-a both in vitro and
in vivo has been reported to be associated with a
functional TNF-a gene polymorphism, consisting
of a guanine (G) to adenine (A) substitution at
Recruitment of subjects
A total of 557 consecutive patients attending the
Scottish Sleep Centre with symptoms of OSAHS
and an apnoea–hypopnoea index (AHI) o15
were asked to participate in the study as index
EUROPEAN RESPIRATORY JOURNAL
VOLUME 26 NUMBER 4
CORRESPONDENCE
R.L. Riha
Respiratory Medicine
Royal Infirmary of Edinburgh
51 Little France Crescent
Edinburgh
EH16 4SA
UK
Fax: 44 1312421776
E-mail: [email protected]
Received:
November 15 2004
Accepted after revision:
July 01 2005
European Respiratory Journal
Print ISSN 0903-1936
Online ISSN 1399-3003
c
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TNF-a AND SLEEP APNOEA
R.L. RIHA ET AL.
cases, and were requested to contact their siblings to ask them
to complete a sleep questionnaire, take part in a sleep study
and provide a blood sample. Out of 312 index subjects willing
to participate in the study, 155 had siblings who were also
available. Some 44 siblings defaulted on attendance and eight
sibling pairs had technically inadequate data collected. In total,
103 sib-pairs (206 subjects) successfully completed the study.
All subjects were Caucasian.
Data collection
After giving written, informed consent, all subjects had their
height, weight and neck circumference and blood pressure
measured, and were asked to fill out a standard sleep
questionnaire (including an Epworth Sleepiness Score (ESS)
[12]). Subjects had 20 mL of blood collected. All siblings had
overnight polysomnography (PSG) using standard techniques
[13]. Studies were performed in sound-proofed and electrically
screened bedrooms at the Edinburgh Sleep Centre (UK).
Sleep was recorded using electroencephalography (EEG),
electro-oculography (EOG) and electromyography (EMG),
using bipolar signals from silver chloride surface electrodes.
EEG was recorded from two scalp sites (Cz/Pz). Frontal EEG
was also recorded using ‘‘mixed’’ channels comprising EEG/
EOG signals (Cz/Fp1, Cz/Fp2). The EOG was recorded from
electrodes placed at sites on the outer canthus of the eye and
Fp1 and Fp2. Submental EMG was recorded using two
electrodes placed on the belly of the genioglossus. A grounding electrode was placed at Fpz. Electrodes placed on the right
and left anterior tibialis recorded leg EMG. All signals were
recorded on the Compumedics W-series and R-series systems
(Compumedics, Melbourne, Australia).
Index patients had either overnight PSG (87 out of 103) or a
home (16 out of 103 cases) study using the limited sleep study
system Edentrace (EdenTec Model 3711 Digital Recorder;
Nellcor, Eden Prairie, Minnesota, USA), which has been
extensively validated against PSG [14, 15]. This device records
oronasal airflow using a thermocouple, chest wall movement
by electrical impedance, heart rate using an electrocardiogram
and finger pulse oximetry; no EEG is recorded.
Sleep studies were analysed by one researcher (R.L. Riha) after
blinding of data using standard scoring criteria [14, 16, 17].
Phenotyping
OSAHS phenotype was defined using AHI and sleepiness, as
measured by the ESS. Table 1 shows the AHI cut-off values
based on normative data for Caucasian subjects [2, 18, 19].
Each subject’s AHI was first scored as either definitely
abnormal, indeterminate or definitely normal on the basis of
sex and age. Each subject’s ESS (out of a total of 24) was then
scored as either sleepy (ESS o11 out of 24) or not abnormally
sleepy (ESS ,11 out of 24). The ESS cut-offs were based on
normative data derived for Caucasian subjects [20, 21].
OSAHS was then classified as being definitely present,
indeterminate or definitely absent by the algorithm represented in table 2.
Blood donors
DNA from UK Caucasian human random control DNA panels
(Product No. HRC-1 96 array and No. HRC-2 96 array;
674
VOLUME 26 NUMBER 4
TABLE 1
Classification of apnoea–hypopnoea index (AHI)
according to sex and age
AHI?h-1
Male
Female
20–40 yrs
,10
,10
40–60 yrs
,15
,10
.60 yrs
,20
,15
20–40 yrs
10–15
10–15
40–60 yrs
15–20
10–15
.60 yrs
20–30
15–20
Normal
Indeterminate
Abnormal
20–40 yrs
.15
.15
40–60 yrs
.20
.15
.60 yrs
.30
.20
TABLE 2
Presence or absence of obstructive sleep
apnoea–hypopnoea syndrome (OSAHS)
phenotype using scores derived for apnoea–
hypopnoea index (AHI) and sleepiness
Score for AHI
Score for sleepiness
OSAHS phenotype
Abnormal
Abnormal
OSAHS
Abnormal
Normal
Indeterminate
Indeterminate
Abnormal
OSAHS
Indeterminate
Normal
No OSAHS
Normal
Abnormal
No OSAHS
Normal
Normal
No OSAHS
European Collection of Cell Cultures, http://www.ecacc.
org.uk) was used as a second set of controls. All donors gave
written informed consent for their blood to be used for
research purposes.
Allelic discrimination analysis
DNA for recruited subjects was extracted using the WizardH
R Genomic DNA Purification Kit (TM050; Promega,
Southampton, UK) or the Nucleon Extraction and Purification Protocol (for extraction of DNA from 10 mL of whole
blood; Nucleon BACC3 RPN 8512; Amersham plc, Little
Chalfont, UK).
The TaqMan system was used to study the -308 (A–G) single
nucleotide polymorphism (SNP). Assay-by-DesignH (Applied
Biosystems, Foster City, CA, USA) was used to design the
probe and primers.
Each 10 mL of PCR contained 10 mg of genomic DNA, 900 mM
primers, 250 mM probes and 2.5 mL of TaqMan Universal PCR
master mix (Applied Biosystems). The solution was pipetted
into each well of a 96- or 384-well plate using an automated
liquid handling robot.
EUROPEAN RESPIRATORY JOURNAL
R.L. RIHA ET AL.
TNF-a AND SLEEP APNOEA
Amplification was performed using the Peltier Thermal Cycler
(PTC-225 DNA tetrad PCR machine; MJ Research, Waltham,
MA, USA). Fluorescence in each well was measured before and
after PCR using an ABI PRISM 7900HT Sequence Detector
(Applied Biosystems) and reproduced as scatter diagrams.
Alleles were read and classified on the plots by two readers
independently blinded to subject status.
Statistical analysis
Between-group comparisons were performed using the Chisquared test and unpaired and paired t-tests. A logistic
regression model using generalised estimating equations was
used to test the association of OSAHS with TNF-a carriage on
an allele-counting basis (assuming Hardy-Weinberg equilibrium and a multiplicative risk model) [22]. The significance
of frequency differences of OSAHS between genotypediscordant sibling pairs was estimated using one sibling
pair from each family by McNemar’s test of symmetry.
Association and linkage analysis of OSAHS with the TNF-a
(-308) SNP was performed using formulae in the SIBASSOC
programme [23]. SIBASSOC performs a Chi-squared test
using the most genotypically distinct unaffected sibling as
a control for each case. The FBAT [24] programme was
used to examine the association of quantitative traits with
genotype. Power was determined using the formulae
described by MACHIN et al. [25] for case-controlled studies
comparing two proportions and using the odds ratio (OR).
The Hardy-Weinberg equilibrium was tested using the
following formula:
1~p2 z2pqzq2
TABLE 3
No OSAHS
Subjects n
Sex ratio M:F
All p-values are for two-tailed tests and considered to be
significant at the a50.05 level.
RESULTS
Sixty-three subjects had no OSAHS and 40 were classed as
intermediate for OSAHS by criteria determined a priori
(tables 1 and 2). Population characteristics for the study
subjects with definite OSAHS and definitely no OSAHS are
presented (table 3). Subjects classified as indeterminate for
OSAHS (n540) were not included in subsequent analyses.
Genotyping results for the -308 (A–G) SNP for TNF-a were
available for 190 UK blood donor controls (99%) and 200 out of
the 206 study subjects (97%). Of the blood donor controls, 95
(50%) were male and the mean¡SD age was 38¡8 yrs.
The distribution of allelic frequencies and genotypes was
significantly different between subjects with a definite diagnosis of OSAHS and UK population controls (table 4). Allele
distribution in the control group (n5190 blood donors) was in
Hardy-Weinberg equilibrium.
Definite OSAHS
63
103
33:30
83:20
p-value
,0.0001
Age yrs
51¡10
52¡9
BMI kg?m-2
27¡5
30¡6
,0.0001
,0.0001
Neck circumference cm
0.6
37¡4
41¡4
SBP mmHg
130¡17
138¡18
DBP mmHg
82¡12
84¡12
0.6
Sleep efficiency#
76¡12
73¡15
0.1
REM time min
70¡24
64¡30
0.2
287¡47
275¡62
0.2
Sa,O2 % awake
97¡2
96¡2
0.07
Lowest Sp,O2 %
90¡7
83¡10
,0.0001
NREM time min
0.007
OSAHS: obstructive sleep apnoea–hypopnoea syndrome; M: male; F: female;
BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood
pressure; REM: rapid eye movement sleep; NREM: nonrapid eye movement
sleep; Sa,O2: arterial oxygen saturation awake; Sp,O2: arterial oxygen saturation
asleep. #: as a percentage of the total time spent asleep divided by the time of
the study.
TABLE 4
ð1Þ
for allelic distribution in the population, and the frequencies
of minor and major alleles were compared with published
frequencies.
Characteristics of the recruited subjects
Allelic and genotype frequencies for tumour
necrosis factor-a (-308) A–G polymorphism in
subjects with definite obstructive sleep apnoea–
hypopnoea syndrome (OSAHS) versus
population controls
Controls
Subjects n
Definite OSAHS
190
103
G/G
130 (69)
52 (51)
A/G
52 (27)
44 (43)
A/A
8 (4)
7 (7)
Alleles n
380
206
G
312 (82)
148 (72)
A
68 (18)
58 (28)
p-value
0.01
0.004
Data are presented as n (%), unless otherwise stated.
of OSAHS or with genotype (p50.4 and p50.5, respectively).
This was based on 35 informative sibling groups.
Forty-five pairs of siblings (males and females) were discordant for the diagnosis of OSAHS (the remainder was
concordant for the diagnosis or one of the siblings had
intermediate phenotype). There was a significantly higher
prevalence of the -308A allele in the siblings with OSAHS
(p50.04) using SIBASSOC. Anthropometric and sleep variables in this group are shown in table 5.
Analysis of quantitative traits (AHI and body mass index
(BMI)) showed no association independently with a diagnosis
Several papers have suggested that there may be sexual
dimorphism in innate immune responses, including the secretion of TNF-a [26–28]. Logistic regression analysis corrected for
sex still showed significant association of the -308A allele with
OSAHS (1.64 (1.06–2.55); p50.023). Further analysis was undertaken using McNemar’s test in 21 pairs of male siblings
EUROPEAN RESPIRATORY JOURNAL
VOLUME 26 NUMBER 4
Logistic regression showed a significant association for TNF-a
(-308A) allele carriage with OSAHS (OR (95% confidence
interval) 1.82 (1.18–2.75); p50.006).
675
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TNF-a AND SLEEP APNOEA
TABLE 5
R.L. RIHA ET AL.
Characteristics of the 45 sibling pairs discordant
for a diagnosis of obstructive sleep apnoea–
hypopnoea syndrome (OSAHS)
No OSAHS
Sex ratio M:F
OSAHS
p-value
,0.0001
23:22
40:5
Age yrs
51¡10
51¡9
BMI kg?m-2
27¡5
30¡6
0.08
Neck circumference cm
37¡4
42¡3
,0.0001
0.6
Sleep efficiency#
77¡10
72¡15
0.05
REM time min
73¡21
65¡32
0.26
Lowest Sp,O2 %
91¡4
82¡10
,0.0001
M: male; F: female; BMI: body mass index; REM: rapid eye movement sleep;
Sp,O2: arterial oxygen saturation asleep. #: sleep efficiency as a percentage of
the total time spent asleep divided by the time of the study.
discordant for carriage of the -308A allele. Fifteen subjects in this
group who were A-allele positive had OSAHS whilst their
siblings did not (p50.001). There was no significant difference
between the 21 sib-pairs with and without the -308A allele with
regard to BMI (28¡3 versus 28¡5 kg?m-2) or age (50¡9 versus
51¡9 yrs). Female sib-pairs in the study were either concordant
for OSAHS or one of the pair had an intermediate phenotype,
precluding similar analysis.
DISCUSSION
In this study, the TNF-a (-308A) allele was significantly
associated with a diagnosis of OSAHS, both in cases versus
random population controls and in siblings. The marked
association between the TNF-a (-308A) allele and OSAHS in
the male sib-pairs discordant for OSAHS strongly suggests that
this relationship is significant. However, this allele is not
required for the development of OSAHS, as its prevalence was
28% in the OSAHS group overall compared with 18% in
population controls.
localised inflammation in the oropharynx leading to increased
cytokine induction, and increased visceral adiposity [30].
In the present study, an independent association of the TNF-a
(-308A) SNP with the diagnosis of OSAHS has been demonstrated. This suggests a disease-promoting role, manifest as
increased susceptibility to symptoms (somnolence and fatigue)
and exacerbation of upper airways inflammation. A further
extension of this work would include measurement of serum
levels of circulating TNF-a, serum-soluble TNF receptor p55
and serum-soluble receptor p75 in all subjects, which the
current study design precluded.
The strengths of this study include the sibling pair design with
additional population controls, the scoring of all records blind
to case or allele status and the a priori agreed classification of
case status. The study has several potential limitations. AHI
was measured on a single night only, when it may vary from
night to night [32]; however, a 1-night study is the standard
and only realistic way of assessing sleep-disordered breathing
in volunteer (or patient) studies. All study participants were
investigated in the same laboratory, using the same standardised techniques for setting up, recording and scoring events.
There was a high intra- and inter-rater reproducibility for
analysing results, which were performed blind to subject
status. The a priori use of an indeterminate score reduced
misclassification by making a wide distinction between those
with definite sleep apnoea and those definitely without it. The
ESS allowed for appraisal of somnolence in daily life
situations, which more objective tests may not [12]. It is the
simplest, most practical and most widely validated and
utilised test for general clinical and research use.
Furthermore, sleepiness is the major presenting symptom of
OSAHS [33].
Inflammation in OSAHS is common and includes elevated
levels of circulating pro-inflammatory cytokines. TNF-a and
interleukin (IL)-6 are elevated in OSAHS, independently of
obesity, and the circadian rhythm of TNF-a secretion is
disrupted [29]. Additionally, other mediators of inflammation
are also elevated, including intracellular adhesion molecule-1
and C-reactive protein [30]. TNF-a, C-reactive protein and IL-6
appear to produce their harmful effects by inducing endothelial dysfunction. TNF-a damages endothelial cells, causes
apoptosis of these cells and triggers procoagulant activity
and fibrin deposition. TNF-a also enhances the production of
reactive oxygen species, including inducible nitric oxide, and
decreases myocardial contractility in a dose-dependent fashion
[31].
Although population stratification is a possible explanation
for the observed association between OSAHS and TNF-a, a
number of factors reduce the likelihood of this. Genotype
frequencies for the non-OSAHS group were in HardyWeinberg equilibrium, mitigating against genotyping or significant population stratification errors. Following PRITCHARD
and ROSENBERG [34], who argued for the use of unlinked genetic
markers to detect population stratification in association
studies, two polymorphisms (serotonin receptor 2A T102C
and growth hormone receptor +561G/T), not known to be in
linkage disequilibrium with the TNF gene, were examined
for association with OSAHS. Genotypic distributions in the
entire group were in Hardy-Weinberg equilibrium and no
associations with OSAHS were detected. However, the
overall number of markers available was insufficient to
carry out a formal test for population stratification.
Numerous studies have also looked at whether stratification
really is a major issue in epidemiological studies and found
the error rates to be extremely small [35, 36].
Sleep disruption in OSAHS may be one factor driving the
increased susceptibility to cardiovascular diseases in this
condition. A genetic propensity towards increased TNF-a
production may further exacerbate these effects. The driving
forces leading to elevations of pro-inflammatory cytokines in
OSAHS may be related to increased activity of both branches
of the autonomic nervous system, intermittent hypoxia,
TNF-a allele frequencies for the normal subjects in this study
were not significantly different compared with published UK
data for Caucasians, excluding bias based on ethnicity [37]. The
power of the study was also adequate to determine a difference
in allelic frequency (two-group continuity corrected Chisquared test of equal proportions showed power of 99% at
a50.05 level) for the analysis between subjects with OSAHS
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R.L. RIHA ET AL.
and blood donor controls. Although the OSAHS status of the
latter group was unknown, this would tend to dilute the
reported difference (by inclusion of OSAHS cases as normals)
rather than strengthen it. Furthermore, blood donors in the UK
are screened to be in good health and the taking of most
medicines prohibits donation. Finally, lack of an independent
association of genotype with quantitative traits for OSAHS
using FBAT may be a reflection of low power in this
population with respect to this statistical method per se.
TNF-a (-308A) is in linkage disequilibrium with human
leukocyte antigen (HLA) class I and II alleles, the class III
region which encodes several components of the complement
system and the major histocompatibility class IV cluster, which
includes lymphotoxin-a (one of five microsatellites within the
TNF locus) and lymphotoxin-b [38]. The association of TNF-a
(-308A) with OSAHS may, therefore, be due to the direct
influence of the SNP in question and/or due to linkage
disequilibrium with other polymorphisms within the TNF-a
gene or other genes within the HLA system.
In conclusion, this is the first known study to examine the
tumour necrosis factor-a -308 (A–G) single nucleotide polymorphism in subjects with obstructive sleep apnoea–
hypopnoea syndrome. The increased prevalence of the -308A
allele in subjects with this disease lends support to the
argument that it is associated with inflammation. Further
research is needed to explore the utility of pro-inflammatory
cytokine gene polymorphisms in obstructive sleep apnoea–
hypopnoea syndrome for risk stratification, thereby paving the
way for potential therapeutic strategies, such as tumour
necrosis factor-a monoclonal antibodies
ACKNOWLEDGMENTS
Helpful assistance with this study was provided by the staff of
the Dept of Sleep Medicine, the Genetics Core Wellcome Trust
Clinical Research Facility (both Edinburgh, UK) and K.
Matthews (statistics).
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